Synthesis,Characterization And Biological Functions Of Organic Trace Elements:Selenium,Iron And Copper

Trace elements play an irreplaceable role in the growth and development of animals by participating in physiological activities such as immune regulation,blood transport,and protein formation.In the feed,the traditional inorganic trace elements supplement is low cost and the synthesis process is simple.Meanwhile,the inorganic trace elements supplement has some disadvantages,including poor palatability,low availability,and environmental pollution.Recently,some organic trace elements chelated by protein or amino acid have been developed,which possess various advantages,such as low toxicity,high bioavailability,and environmentally friendly.However,the organic trace elements are difficult to be applied in the breeding process due to their complex production process and high cost.In this study,Bacillus licheniformis CGMCC 2876,a microbial feed additive was selected to produce the extracellular polymeric substance（EPS）for the preparation of novel organic trace elements.Furthermore,the physicochemical properties of these organic trace elements were characterized and their biological functions were explored.It was expected to lay a theoretical and practical foundation for the application of the novel organic trace elements in animal breeding.The main research results are as follows:（1）Synthesization of organic trace elementsEPS was obtained by B.licheniformis CGMCC 2876 with industrial waste sugarcane molasses as the sole carbon source.The highest yield of EPS could reach 5.7g/L,when the concentration of sugarcane molasses was 80 g/L and the fermentation time was 56 h.Then,EPS and sodium selenite（Na₂SeO₃）were used to synthesize organic extracellular polymeric substances-selenium nanoparticles（C-EPS-Se NPs）in vitro with ascorbic acid as a reducing agent.The diameter of C-EPS-Se NPs was 141.8nm and the content of Se in C-EPS-Se NPs was 107.43 mg/g.Organic iron（EPS-Fe）was synthesized from EPS of B.licheniformis CGMCC 2876 and soluble metal salt ferric chloride（Fe Cl₃）by complexation reaction.The optimum synthesis conditions of EPS-Fe were explored through the single factor optimization experiment.The Fe content of EPS-Fe could reach 77.4 mg/g when the mass ratio of EPS to trisodium citrate was 2:1,the reaction temperature was at 70℃,and the reaction p H was 8.5-9.Organic copper（EPS-Cu）was synthesized from EPS of B.licheniformis CGMCC2876 and soluble metal salt copper chloride by complexation reaction.The blue EPS-Cu was synthesis under the best conditions of EPS-Fe synthesis,and the content of Cu in EPS-Cu could reach 90.67 mg/g.In addition,inorganic Se could also be transformed into organic Se by adding Na₂SeO₃ in the medium of B.licheniformis CGMCC 2876.The organic Se can be found in the bacterial cells and EPS,with a Se concentration of82.4 mg/g and 4.72 mg/g,respectively.（2）Characterization of organic trace elementsThe physicochemical properties of organic trace elements was charactered by UV spectrum,IR spectrum,transmission electron microscope and X-ray electron energy spectrum and so on.The zero-valence Se by C—O···Se bonds or through hydrogen bonding interaction（O—H···Se）asymmetric stretch were observed in C-EPS-Se NPs.In addition,the aqueous solution of C-EPS-Se NPs exhibited good stability,and the C-EPS-Se NPs were resistant to the digestion of simulated gastric fluid.In EPS-Fe,the Fe²⁺and Fe³⁺was found,and Fe were complexed with EPS through Fe—OOH bonds or Fe—O bonds.EPS-Fe in the potassium ferrocyanide solution was the same as in the distilled water as a clear transparent brown solution,indicating that EPS-Fe did not contain free of Fe³⁺,proving its high stability.In EPS-Cu,Cu ²⁺and Cu⁰ was found,and Cu were complexed with EPS through Cu—O bonds.（3）Investigation of biological functions of organic trace elementsBiological functions of organic trace elements were investigated.It was found that the cytotoxicity of organic trace elements was lower than that of inorganic trace elements.C-EPS-Se NPs and EPS-Fe with concentration under 800μg/mL had no cytotoxicity.EPS-Cu with concentration under 50μg/mL had no cytotoxicity.C-EPS-Se NPs,EPS-Fe and EPS-Cu showed the scavenging ability towards·OH,DPPH and·O^2-,and they could protect cells from hydrogen peroxide damage in the range of500μg/mL,500μg/mL and 50μg/mL,respectively.When the concentration of organic trace elements were 1000μg/mL,they all had inhibitory effect on gram-positive and gram-negative（opportunistic）pathogens.When the concentration of C-EPS-Se NPs and EPS-Fe were 800μg/mL,they also could inhibit the proliferation of H22 cells,EPS-Cu inhibited the proliferation of Hep G2 cells.In this study,EPS were produced by B.licheniformis CGMCC 2876 using industrial waste sugarcane molasses as the sole carbon source.And the EPS could be further used to prepare organic Se,Fe,and Cu,which could not only improve the value of waste biomass but also reduce the production cost of organic trace elements.In addition,the organic trace elements possess the biological functions of both EPS and trace elements,including non-toxic and anti-oxidation,anti-pathogen,and anti-cancer cell proliferation abilities.Thus,the organic trace elements synthesized in the present study are expected to become a new generation of trace element additives used in animal feed,to promote the sustainable development of the breeding industry.